The overall goal of the proposed research is to develop, through appropriate manipulations of a suitable attenuated Salmonella typhi live vector, a mucosally-administered multivalent vaccine to prevent diphtheria, pertussis, and tetanus (i.e., a mucosal DTP vaccine). To accomplish this, the PI will initially successfully express within attenuate S. typhi appropriate protective antigens from C. diphtheriae, B. pertussis, and C. tetani and confirm, using the murine intranasal model of immunogenicity, that such constructs elicit the relevant types of immune responses. The PI and his colleagues will then attempt to improve such immune responses in several ways: (1) relevant proteins will be expressed in attenuated S. typhi strains in which expression of the Vi capsular polysaccharide has either been removed or expression has been made constitutive. (2) A fusion protein consisting of fragment C of tetanus toxin fused to the truncated S1 subunit of pertussis toxin, used as a test antigen, will be secreted extracellularly using the E. coli hemolysin and secretion apparatus. (3) Proteins will be expressed from stabilized plasmids which encode a critical enzyme necessary for survival of the attenuated S. typhi carrier strain. (4) The fragment C-S1 fusion protein will be expressed in S. typhi live vector strains which will co-express either a mutant heat-labile enterotoxin of E. coli (the K63 LT holotoxoid) that functions as a powerful adjuvant yet does not cause intestinal secretion or will co-express the IL-4 cytokine (which has the effect of enhancing antibody responses). The PI and his colleagues will make amino acid substitutions in the NAD-binding region of diphtheria toxin, aiming to construct a stable mutant that lacks enzymatic (i.e., toxic) activity, but retains the ability to stimulate neutralizing antitoxin. It is expected that by the end of the research plan, all the individual constructs necessary to stimulate protective immune responses will have been constructed, their immunogenicity established in the mouse intranasal immunization model and modifications selected to enhance the specific immune responses. This will set the stage for a future effort that would examine the immunogenicity of a prototype multivalent DTP vaccine consisting of a mixture of the optimized CVD908-htrA constructs expressing diphtheria, pertussis and tetanus antigens.